THE EFFECT OF BTK INHIBITION ON PLATELET ACTIVATION BY GPVI AND CLEC-2 by Phillip Lindsay Ross Nicolson A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Cardiovascular Sciences College of Medical and Dental Sciences University of Birmingham October 2018 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Background The novel Btk inhibitor ibrutinib has recently been introduced into the clinic for treatment of lymphoproliferative disorders (LPDs) where it is unexpectedly associated with major bleeding. It has been shown to block GPVI and CLEC-2- mediated platelet function with the former correlating with bleeding. This contrasts with patients who are deficient in GPVI or Btk who experience minor/no bleeding respectively. Mice that lack CLEC-2 also have no bleeding diathesis but are protected from thromboinflammation. Aims This study aims to investigate the discrepancy between the bleeding seen with ibrutinib and those that lack Btk. It also aims to examine whether ibrutinib can block CLEC-2 signalling in mouse and human venous thrombosis. Results Ibrutinib blocks GPVI signalling due to off-target effects which occur because of the high clinical doses that are used. It blocks CLEC-2 signalling at ~50-fold lower concentrations and reduces venous thrombosis rates in patients taking it for LPD. Conclusions Lowering the dose of ibrutinib or developing a more specific Btk inhibitor would ameliorate the major bleeding that is seen with ibrutinib. Btk inhibitors are suitable for further study as inhibitors of CLEC-2 signalling in human venous thrombosis. Publications arising from this thesis Manuscripts • Nicolson PLR, Hughes CE, Watson S, Nock SH, Hardy AT, Watson CN, Montague SJ, Malcor JD, Thomas MR, Tomlinson MG, Pratt G, Watson SP. Inhibition Of Btk By Btk-Specific Concentrations Of Ibrutinib And Acalabrutinib Delays But Does Not Block Platelet Aggregation To GPVI. Haematologica. 2018;103(12):2097-2108 DOI: 10.3324/haematol.2018.193391 Book Chapter • Haining EJ, Nicolson PLR, Onselaer MB, Poulter NS, Thomas MR, Rayes J and Watson SP. (2019). ‘GPVI and CLEC-2’ in Michelson AD (ed.) Platelets, 4th edition. Elsevier/Academic Press, pp 213-226. Dedication I would like to dedicate this thesis to my wife Pippa and my children William and Emily without whom I would not have achieved the spiritual, physical and psychological nourishment necessary to sustain me throughout this degree. Acknowledgements This work was supported by the University of Birmingham Institute of Cardiovascular Sciences and the Institute of Translational Medicine as well as the British Heart Foundation through the clinical research training fellowship (FS/17/20/32738). I would particularly like to acknowledge Lorraine Harper for enabling me to get on the first rung of the ladder to start this work. First and foremost for help in almost every aspect of laboratory work I want to thank the incredible Stef Watson without whom numerous experiments would have failed due to lack of expertise and reagents. Practical help with other aspects of laboratory work was received from Craig Hughes and Alex Hardy for the aggregation and biochemical assays; from Alex Bye and Jon Gibbins for the 2+ Ca mobilisation assay; from Vicky Simms and Steve Thomas for the flow adhesion assays; from Abs Khan, Natalie Poulter, Jeremy Pike and Dee Kavanagh for the spreading and super-resolution microscopy assays and Lizzie Haining, Julie Rayes and Alex Brill as well as all the staff at the Biomedical Services Unit for the animal work. Social and gastronomic support in the lab was obtained through close collaboration with Abs Khan, Natalie Poulter, Chris Smith and Pushpa Patel. For help with collection and provision of patient blood I would like to thank Mark Crowther, Nick Pemberton, Salim Shafeek, Kate Arthur, Gaynor Pemberton, Lesley Candlin and Rebekah Hart at Worcestershire Royal Hospital, Shankara Paneesha, Aarnoud Huissoon, Hayley Clifford, Alison Hardy and Melanie Kelly at Birmingham Heartlands Hospital and Guy Pratt, Tina McSkeane, Gillian Marshall and Michelle Harry at the Queen Elizabeth Hospital. For help with collection of human blood and tissue and collaboration on the same I would also like to thank Rachel Brown from the Queen Elizabeth Hospital, Jane Steele and Gareth Bicknell from the Human Biomaterials Resource Centre and John Welsh and Mark Kahn from the University of Pennsylvania in Philadelphia. I would also like to thank Andrew Wilkinson and Robert Neely from the School of Chemistry at the University of Birmingham for their help with the chemical analysis of ibrutinib and acalabrutinib. For working so diligently under my supervision and providing work to go towards the conclusions of this thesis (even if not directly examinable) I want to thank Josh Hinds. In particular for help with statistical analysis for fellowship applications and work included in this thesis I would like to thank Peter Nightingale from the Queen Elizabeth Hospital for so willingly giving up his time for me. I would also like to thank Gill Lowe, Guy Pratt, Gayle Halford and Mike Tomlinson for helping me learn to cope with Steve Watson. The gratitude for this also extends to every other member of the Birmingham Platelet Group. Outside the lab I would like to thank my parents and parents-in-law for their ongoing support with childcare to enable conference travel and unsociable working hours. I would also like to thank my good friends Ian Butler and Parag Gajendragadkar for forcing me out on my bike to the Clent hills and Italian Dolomites respectively. I would like to thank Iain Johnston, Alessandro Di Maio, Matt MacKenzie and Abs Khan for similarly dragging me out to music rehearsal studios in Digbeth. I would also like to thank Dave Grohl, Nate Mendel, Taylor Hawkins and Chris Shiflett as well as Shawn Crahan, Craig Jones, Mick Thomson, Corey Taylor, Sid Wilson, Chris Fehn, Jim Root, Alessandro Venturella, Jay Weinberg, Joey Jordison, Paul Gray and Donnie Steele for fuelling the thesis writing process. Finally I would like to thank my supervisors, Steve Watson and Guy Pratt, for their unfailing support before and throughout this work. For many practical, financial and psychological reasons, without Steve, none of what follows would have been possible. I also want to thank him for bringing me down a peg or two at the times when my confidence outweighed my abilities; “Pip, in science, you never prove anything”. Table of Contents CHAPTER 1: GENERAL INTRODUCTION ................................................................................. 1 1.1 Introduction to platelets ........................................................................................................ 1 1.1.1 Platelet production ........................................................................................................ 1 1.1.2 Platelet structure ........................................................................................................... 2 1.1.3 Platelet function ............................................................................................................. 3 1.1.3.1 The role of platelets in haemostasis and thrombosis ................................................. 3 1.1.3.2 Functions of platelets outside haemostasis ............................................................... 5 1.1.4 Major platelet receptors and ligands ............................................................................. 6 1.1.4.1 GPCRs ....................................................................................................................... 7 1.1.4.2 ITAM receptors ........................................................................................................... 8 1.1.4.3 Adhesion receptors .................................................................................................. 10 1.2 Haemostasis ...................................................................................................................... 11 1.2.1 Classical Haemostasis ................................................................................................ 11 1.2.2 Inflammatory haemostasis .......................................................................................... 13 1.3 Platelets in thrombosis and thromboinflammation ............................................................. 14 1.3.1 Arterial thrombosis ...................................................................................................... 14 1.3.2 Venous thrombosis ..................................................................................................... 15 1.3.3 Immunothrombosis and thromboinflammation ............................................................ 18 1.3.4 ITAM receptors as new targets in thrombosis and thromboinflammation ................... 19 1.4 Bruton’s tyrosine kinase (Btk) and its inhibitors ................................................................. 21 1.4.1 Btk ..............................................................................................................................